Whole genome sequencing of DNA from subjects at high risk for cancer predisposition has led to the identification of numerous variants of unknown significance, VUSs. We evaluated 48 women from high-risk families diagnosed with ovarian cancer for germline mutations by whole exome sequencing of their germline DNA. These subjects were previously found to be negative for any known pathogenic mutations in either BRCA1 or BRCA2. Numerous genetic variants with a moderate to high effect in relevant pathways were found to be present in this cohort. In particular, we identified 2 deleterious and 5 potentially damaging variants in the ATM gene in the normal DNA from these high-risk women with ovarian cancer. The role of ATM in ovarian cancer has been underestimated because of a lack of functional assays for the effects of these genetic changes. In this project we will pilot the development of a general approach to determine the nature of private and rare mutations in DNA repair associated genes using cell-based functional assays. The functional analysis of novel germline variants in the gene ataxia-telangiectasia mutated (ATM) will be studied in immortalized normal ovarian and mammary epithelial cells. This approach is superior to using established tumor cells that already have DNA repair defects. The PI's lab is experienced in studying the genetics of cancer and the ramifications of mutant hereditary cancer genes on normal cell biology and genomic instability focusing previously on Li-Fraumeni Syndrome. ATM codes for a protein kinase that regulates the DNA damage response initiating the signaling cascades that activate cell cycle checkpoints and the repair of DNA double-strand breaks. We will develop cell-based assays that can be used for the functional analysis of genetic variants of unknown significance (VUSs) in the ATM gene. ATM mutations are associated with the risk of breast cancer; however little information is available on ovarian cancer. Therefore, we will compare the biological and biochemical impact on immortalized normal mammary and ovarian epithelial cells that have had their ATM genes edited to contain the SNPs that we have identified in patients' DNA. We will determine whether these clearly deleterious and potentially damaging variants have significant impact on cell cycle or DNA repair mechanisms in a cell type specific fashion. Deleterious and potentially damaging VUSs in ATM were statistically overrepresented in our cohort (Table 1) compared to the MAF in the 1000 genomes database. However each VUS will require some functional analysis to provide clinically useful information to patients. This project will be pursued in two aims: AIM 1: We will employ gene editing of the endogenous ATM gene in hTERT immortalized normal mammary and ovarian epithelial cells using CRISPR/Cas9 to produce potentially deleterious ATM variants. AIM 2: Using functional assays in pathways known to be associated with inherited risk of ovarian cancer we will determine the impact of these VUSs on cell cycle control and DNA repair mechanisms.